The 5' leader region of retroviral genomic RNA, while not translated, is replete with other non-coding functionality, including sequences important for transactivation of transcription, primer binding, splicing, encapsidation, dimerization of viral RNA, and initiation of translation of the gag ORF. Within a species, phylogenetic conservation of these non-coding sequences rivals even that of conserved coding domains for the viral proteins. This observation emphasizes that the sequence and structure of the leader region are essential for viral replication. The high degree of conservation also highlights the potential to target this region for antiretroviral strategies. The use of conserved RNA sequences as drug targets have been well established, since many ribosome-targeting antibiotics recognize a specific segment of bacterial rRNA. Recent in vitro results have demonstrated that structure of the leader RNA is plastic by design and its multiple conformations appear to give rise to functions appropriate to the stage of the replication cycle. In the last grant period, we elucidated structural and dynamic aspects of the leader region of the genomic RNA of HIV-2, one of the causative agents of AIDS in humans. In particular, while searching for signals important for genomic RNA dimerization, we discovered that these signals were interspersed with and overlaped with known signals for encapsidation, splicing, and translation of viral RNAs. Based on the in vitro characterization of conformational isomers of HIV-2 leader RNA and upon the high degree of phylogenetic conservation of involved sequences, we hypothesize that the leader region of HIV-2 and other retroviruses is intimately involved in the regulation of several viral processes, and that this regulation is manifested by differential presentation of the signaling structures found in the leader region. In this application we propose to test this hypothesis by constructing and characterizing viral mutants harboring substitution and deletion mutations designed to interfere with long- and short-range RNA interactions that were shown in vitro to be important in the modulation of RNA structure and behavior. We will use a combination cell culture and biochemical approaches to characterize the largely unexplored interrelationships of RNA dimerization, encapsidation, and translation in HIV-2. The studies proposed here will aid in our overall understanding of the biochemistry and cell biology of HIV-2 replication as well as to validate the 5' leader region RNA as a potential antiretroviral target.